DE69221118T2 - Target component assay - Google Patents

Abstract

An improved assay of target components in a sample utilizes specific gravity-altering particles which are attached to the target components by specific antibodies. The attached specific gravity-altering particles are preferably liposomes which will buoy or sink the targets to a common level in the specimen sample when the latter has been centrifuged in a transparent tube. The liposomes can provide an accentuated and more pronounced indication of the presence of the targets in the sample due to their ability to contain many multiples of fluorescent or non-fluorescent dye molecules with minimal steric interference with the attached antibodies' binding ability. <IMAGE>

Description

This invention relates to assaying target components of biological specimens. More specifically, this invention relates to detecting and/or quantifying target cells, particles or organisms, hereinafter referred to as "targets," in biological specimens by simultaneously altering the specific gravity and highlighting or marking the targets.

U.S. Patent No. 4,181,609, issued January 1, 1980 to S. C. Wardlaw et al., discloses a blood analysis method in which certain blood cells (reticulocytes) are condensed so that a clear cell interface is formed in a centrifuged blood sample. Thus, changing the natural relative density of the reticulocytes results in an improved blood test. U.S. Patent No. 4,332,785, issued January 1, 1982 to Allen et al., discloses a specific method that uses fluorescent antibodies to label reticulocytes in a quantitative analysis of reticulocytes in a blood sample; and US Patent No. 4,591,570 issued to Chang on May 27, 1986 discloses the use of a number of different antibodies spotted on a support to capture a plurality of different antigens in an immunoassay procedure. However, the prior art does not disclose a general method involving altering the relative density of a number of different components of the preparation sample to accumulate the altered components in a centrifuged preparation sample and to label the components to make them easily identifiable. The same applies to EP-A-0 179 007 and EP-A-0 241 042.

EP-A-0 179 007 discloses the removal of cells from a medium by using particles that specifically bind to the target cells, where the particles have a lower relative density than the medium. To quantify the amount of cells bound to the particles, the sample is viewed under a microscope.

EP-A-0 241 042 discloses microcapsules, e.g. liposomes, containing a labeling material, e.g. fluorescent substances or dyes, and having antibodies bound to their surfaces. Cells to be analyzed are combined with microcapsules containing antibodies that react with them. The labeling material allows the optical detection of the resulting immunocomplex.

This invention relates to an improved analysis of target components of preparation samples which involves selectively binding observably different liposomes to the respective target components of the sample. The liposomes are bound to the preparation components by means of antibodies attached to the surface of the liposomes. The antibodies will be at least one antibody specific for a surface antigen known to be present on the target component of the sample. Different antibodies can be bound to the surface of a single liposome simultaneously so that the analysis can be specific for any of many different targets. Differentiation of the liposomes is preferably accomplished by a visible or machine-readable distinguishing label which is enclosed inside the liposomes or incorporated into the phospholipid bilayer. The distinctive marking means may be a visible dye, a machine-readable dye, a radioactive radiation source or the like.

Liposomes are microscopic, spherical, artificial structures that consist primarily of phospholipids. A liposome can consist of one or more lamellar phospholipid vesicles that form a closed spherical shell that can be loaded or filled with a material such as a liquid or the like. Liposomes range in size from 150-250 nm and the average thickness of a lipid membrane is 2.5 nm, it is obvious that the density of the liposome is determined primarily by the density of the entrapped substance, ie, the dye or indicator and the buffer or carrier medium. Methods of making and using liposomes are disclosed in "Liposomes: Diagnostic and Therapeutic Applications" by James O'Connell, in the December 1988 issue of Medical Device and Diagnostic Industry, pages 31-36.

This invention relates to the use of specially prepared liposomes to separate and highlight different components of a specimen sample. The liposomes will be filled or loaded with a marking or highlighting material such as a liquid containing a visible or machine-readable dye or any other detectable component. The filling material will have a predetermined specific gravity which will thus determine the specific gravity of the liposomes. Attached to the outer surface of the liposomes will be one or more different antibodies which will be specific for different surface antigens known to be present at different targets in different samples to be tested. For example, the liposomes could be filled with a fluorescent liquid having a specific gravity of 1.5 and could have attached to them antibodies A, B, C and D which would be specific for surface antigens a, b, c and d. These surface antigens would be antigens known to be present at one or more targets in different specimen samples that could be analyzed qualitatively or quantitatively. In this way, for example, a single analysis medium could be used to analyze several different samples. It is also possible to use different liposomes A-D, each with its own density, to examine the aforementioned surface antigens a-d simultaneously.

From the above general example, it will be seen that the invention has wide applicability in the medical field for diagnostic and/or quantification procedures. One only needs to know the relative density of the component to be examined and what surface antigens it possesses. Once these facts are known, a liposome can be created to label the component and accumulate it in the sample in which it is present. The antibodies used can be polyclonal or monoclonal antibodies.

It is an object of this invention to provide an improved method for testing a preparation sample for a specific target component therein.

It is a further object of this invention to provide a method of the type described which can alter the relative density of the target component and also highlight the target components to make them detectable in the sample.

It is yet another object of this invention to provide a method of the type described which can analyze the target components in the sample quantitatively and/or qualitatively.

The objects are achieved by the methods of claims 1 and 2. Particularly advantageous applications are claimed in claims 3 to 8.

The objects and advantages of the invention will become more apparent from the following detailed description of several preferred embodiments thereof when taken in conjunction with the accompanying drawing which is a schematic view of a liposome modified for use in connection with this invention.

Referring to the drawing, a monolamellar liposome vesicle is shown, generally designated by the numeral 2. The membrane 4 of the vesicle 2 is very thin, about 40 Å thick, yet encloses a relatively large volume. The interior of the vesicle contains a labeling liquid 6 such as a dye or the like, which may be a carrier liquid or dispersed in a carrier liquid. The antibodies 8 are bound to the outside of the membrane 4. As an example, the four different antibodies A, B, C and D are shown on the outside of the membrane 4. As indicated above, in fact, hundreds (or thousands) of different antibodies can be bound to each vesicle if desired. It is believed that the antibodies are mobile over the outside of the vesicle 2, so that no particular orientation of the vesicle is necessary to obtain the desired labeling of the target components. It will be noted that because of the immensely larger proportion of the labeling agent 6 enclosed in the vesicle compared to the membrane 4, the relative density of the labeling agent 6 and/or its carrier determines the relative density of the vesicle 2.

The vesicles 2 can be prepared by any conventional method, in which the labeling agent 6 is included during the preparation of the vesicles, as described in the O'Connell reference given above. Szoka and Papahadjopoulos in their article "Liposomes: Preparation and Characterization", pages 69-82, From Physical Structure to Therapeutic Applications, Elsevier/North Holland Biomedical Press 1981, describe several methods for attaching antibodies to liposomes. For example, the liposomes can be prepared by incorporating 5 mM fluorescein sulfonic acid labeling agent dissolved in 5 mM EDTA buffer vehicle at pH 4.5. Antibodies can be coupled to the intact liposomes via a Schiff base which can be prepared at neutral or alkaline pH according to the method described by Fiddler and Gray in Vol 86 of Analytical Biochemistry at pages 716-724. This method uses periodate oxidation of liposomes containing 10 mole percent lactosylcerebroside or mixed brain gangliosides. The oxidation step is carried out under either acidic (pH 5.5) or alkaline (pH 8.4) conditions. The duration of the periodate oxidation at pH 5.5 must be carefully controlled to prevent periodate from entering the liposomes. A subsequent reduction step with sodium cyanoborohydride is carried out at neutral pH. During the reaction, the integrity of the vesicles is maintained, as indicated by the fact that neither entrapped contents leak out of the liposomes nor entrapped periodate-cleavable components are oxidized. In the above technique, protein coupling is effective. Protein-to-protein cross-linking or liposome aggregation are not serious problems in the above-mentioned process.

This invention can be used in the quantification of reticulocytes in a whole blood sample. Reticulocytes are young red blood cells, and the quantitative measurement of reticulocytes in a blood sample can be used to determine the body's rate of red blood cell production. Reticulocyte quantification is important in determining the cause of anemia, and it can also be used to determine the presence of "compensated blood loss," that is, a normal amount of red blood cells present only because of an extremely high rate of red blood cell production. Such compensated blood loss can be an early sign of the presence of gastrointestinal bleeding due to malignancy or other causes. Reticulocytes have the surface antigen transferrin to which antitransferrin antibodies can bind. Thus, the reticulocyte population of a whole blood sample can be quantitatively determined by binding the antitransferrin antibodies to the membrane of liposomes containing a liquid stain such as a dye or a fluorescent stain having a specific gravity different from that of reticulocytes and mature red cells. The labeling liposomes are then mixed with the blood sample to the extent necessary to bind all of the reticulocytes in the blood sample. The mixture is then placed in a blood sample tube of the invention disclosed in U.S. Patent No. 4,027,660 issued to SC Wardlaw et al. and quantified by the methods described therein.

This invention can also be used to detect and quantify T lymphocytes and their subsets in the blood of an individual. T lymphocytes are a subset of lymphocytes that are mediators of cellular immunity; and B lymphocytes are mediators of humoral immunity (antibody producers).

A discussion of lymphocyte reactivity to specific antigens in blood is contained in U.S. Patent Application No. 07/340,248 filed April 19, 1989 by Robert A. Levine and Stephen C. Wardlaw, which resulted in issued patents No. 5,360,719 and No. 5,480,778. Activated lymphocytes (lymphoblasts) have surface activation antigens such as a transferrin receptor, HLA-Dr; Leu-23, and the like. To detect the lymphoblasts, antibodies specific for one of the aforementioned lymphocyte antigens are bound to liposomes incorporating a suitable labeling agent. The labeling liposomes are then mixed with a blood sample for a time sufficient to allow the liposomes to bind to any lymphoblasts that may be present in the blood sample. The mixture is then drawn into a blood test tube of the type described in U.S. Patent No. 4,027,660 and tested according to the procedures described therein.

Since the cells to be examined are white cells, the labeling dye will preferably be selected at a relative density different from the white cells in order to cause any labeled lymphoblasts to segregate away from the rest of the white cells or to layer in a localized band within the white cells. The liposomes used in lymphocyte subset selection for T lymphocytes have a density of less than 1.017 g/ml. Lymphocytes have a mean density of 1.06 g/ml and a density range of 1.055-1.070 g/ml. Therefore, the liposomes used are capable of reducing the density of the target T lymphocytes, causing them to rise to the top of the lymphocyte layer.

The following is an example of the use of the invention for the detection of T lymphocytes in a blood sample. A labeling agent composition containing 25 µl of undiluted 55-2/LEU-1 antibody coupled to liposomes loaded with a fluorescein (fluorescent) dye having a specific gravity of less than 1.017 g/ml was added to 1 ml of EDTA venous blood, and to this mixture was added 25 µl of undiluted 0.42 g/10 ml stock solution of sodium fluoride. The sodium fluoride produces a sharper separation of the non-fluorescent and fluorescent components of the lymphocyte cell bands. To the above mixture was added 50 µl of undiluted 1.1 g/10 ml stock solution of potassium oxalate to give a sharper red cell-granulocyte separation as described in the first prior art mentioned above. The resulting mixture was allowed to incubate for five minutes after which the mixture was centrifuged to separate the various cell types in a capillary or other transparent tube containing a plastic float that expands the various cells in the sample. Using the above technique, a distinct band of fluorescent lymphocytes was formed in the white cell layer. This band was quantified by measuring their axial extension in the tube. The resulting value was indicative of the T lymphocyte cells circulating in the blood. If dyes or stains with a different specific gravity are used, the labeled cells can be induced to segregate elsewhere in the centrifuged blood sample.

The invention can also be used to analyze other cells, particles or organisms in samples of biological fluids. The presence of abnormal amounts of beta-amyloid protein (BAP) is known to occur in the brain, skin and colon mucosa of patients suffering from Alzheimer's disease, a degenerative neurological disease, and in elderly patients with Down syndrome, a congenital disease also known as trisomy 21. Until now, BAP has not been detectable in serum. The presence of BAP in white blood cells of the lymphocyte type or other types can be detected using liposome-bound antibodies directed against surface antigens on BAP, the antigens being exposed on the surface of the circulating cells that produce BAP.

As far as the detection of organisms in samples of biological fluids is concerned, malaria protozoa are essentially intracellular organisms that are located within red blood cells by immunological means, since antibodies are unable to penetrate the red blood cell membrane in intact living cells. Falciparum-type malaria protozoa produce characteristic red cell changes in infected red blood cells. There is a vital need to distinguish falciparum malaria from non-falciparum malaria, since the former is often fatal and often resistant to commonly used antimalarial drugs. It is difficult for non-specialists to distinguish morphologically between falciparum malaria and non-falciparum malaria. The use of labeling liposomes that can bind to red cells infected with falciparum malaria would enable a technician to identify the infection. The antibodies on the liposomes are specific for a red cell membrane surface antigen that is unique to falciparum-infected red cells.

Claims (8)

1. A method for testing a sample of a biological fluid for the presence of a target component in the sample, comprising the following steps: a) providing a plurality of liposome vesicles having bound to an outer surface of the vesicle an antibody specific for a surface antigen on the target component; wherein the vesicles have incorporated a labeling material and/or carrier material that makes the vesicles detectable in the sample of biological fluid, and wherein the labeling material and/or carrier material imparts to the vesicles a relative density that is different from the relative density of the target component; b) adding the vesicles to the liquid sample to form a mixture thereof; c) centrifuging the mixture to separate any liposome-target combinations into a separate band in the mixture; d) Identifying and/or quantifying the own band in the centrifuged mixture. 2. A method for testing a sample of a biological fluid for the presence of at least two target components in the sample, comprising the following steps: a) providing a plurality of first liposome vesicles having bound to an outer surface of the first vesicles a first antibody specific for a surface antigen on one of the target components; wherein the first vesicles have incorporated therein an first labeling material that makes the first vesicles detectable in the sample of biological fluid, and wherein the first labeling material imparts to the first vesicles a first relative density which is different from the relative density of one of the target components; b) providing a plurality of second liposome vesicles having bound to an outer surface of the second vesicle a second antibody specific for a surface antigen on another of the target components; wherein the second vesicles have incorporated therein a second labeling material that renders the second vesicles detectable in the sample of biological fluid, and wherein the second labeling material imparts to the second vesicles a relative density that is different from the relative density of the other of the target components and that is also different from the first relative density; c) adding the vesicles to the liquid sample to form a mixture thereof; d) centrifuging the mixture to separate any liposome-target combinations into separate bands in the mixture; e) Identify and/or quantify the own bands in the centrifuged mixture. 3. The method of claim 1 or 2, wherein the sample of biological fluid is a blood sample. 4. The method of claim 3, wherein the target component is a specific blood cell type. 5. The method of claim 4, wherein the target component is T lymphocytes or their subsets. 6. The method of claim 4, wherein the target component is reticulocytes. 7. The method of claim 4, wherein the target component is BAP-producing blood cells. 8. The method of claim 4, wherein the target component is falciparum-infected red cells.